Ab181125

Cells were placed on autoclaved 12mm x 12mm glass coverslips in twelve well cell culture plates. The next day, the cells were treated with the indicated drug in separate cell culture wells. 24 hours later, fresh culture media was added for a post-drug recovery period of 24 hours. Later they were fixed with either 4% paraformaldehyde for 15 min at RT (when staining for of STING) or cold (−20 °C) methanol for 5min at RT (when staining for Rel-B). For STING, selective plasma membrane permeabilization was done using 0.02% Saponin in TBS for 2 min. For Rel-B, nuclear permeabilization was done using 0.15% of Triton X-100 in TBS for 10 min. TBS + 1% BSA + 22.52 mg/ml of glycine was used as a blocking agent for 45 minutes. TBS + 1%BSA was used as blocking agent during primary antibody staining (STING at a dilution of 1:1000; Abcam Cat. No. ab181125; RelB at a dilution of 1:500; Abcam Cat. No. ab180127) overnight at 4 °C. After washing the cells, they were incubated with goat anti-rabbit Alexa Fluor 647 (at a dilution of 1:1000; Abcam Cat. No. ab150083). The coverslips were treated with DAPI (0.1ug/ml in TBS) for 2 min. The coverslips were then mounted with Prolong Diamond Antifade Mountant (ThermoFisher Scientific; Cat No., P36965). The cells were viewed on a spinning disk confocal microscope (UltraVIEW Vox; PerkinElmer) and quantified using Volocity version 6.3.